<p>The study focused on preparing, evaluating, and comparing several interfaces consisting of electrochemically seeded gold nanoparticles (AuNPs) on a commercial screen-printed graphite electrode. The seeding process involved two types of biopolymers, chitosan (Ch) and polygalacturonic acid (PGA), to explore their functional groups (-NH₂ and -COOH, respectively) as immobilization interfaces for adsorbing asparaginase (AG) and detecting anti-asparaginase (AB) in physiological fluid. The AB/AG system served as a model to test the assembled biosensor as a potential point-of-care tool for children with Acute Lymphocytic Leukemia (ALL), aiming to provide a less invasive test with quicker responses compared to the Enzyme-Linked Immunosorbent Assay (ELISA). The electrochemical seeding of AuNPs on screen-printed electrodes (SPEs) was presented as an application of a surface prepared using double pulse chronoamperometry, with parameters (pulse number and time) adjusted to ensure multiple detection sites for a low-concentration biomarker like AB. Electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to monitor gradual changes in the AB/AG/AuNPs-biopolymer/SPE interfaces. Scanning electron microscopy (SEM) images provided the size distribution of the deposited nanostructures, an estimation of the active area, and the deposited mass for the physical characterization of the proposed biosensor. Electrochemical impedance spectroscopy (EIS) and fluorescence spectroscopy corroborated the presence of immobilized AG proteins on the nanocomposite, as expected during biosensor fabrication. Fluorescence spectra comparisons indicated that AB recognition on the prepared biosensor corresponded with the impedance results, as the capacitance change of the AB/AG/AuNP-PGA interface aligned with the expected fluorescence on/off behavior.</p> Graphical abstract <p></p>